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Differences in gene expression in embryos seen when frozen semen is used

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  • Differences in gene expression in embryos seen when frozen semen is used

    Things aren't quite as simple as we believed.

    Interesting study that is available on PLoS One here: ] And summarized here:

    Here's the complete discussion section:

    Here we report, for the first time, evidence that procedures performed during handling of sperm, such as freezing and thawing, have a significant impact on critical aspects of the early embryo transcriptome. The equine model used in our study has a number of advantages, including a long pre-attachment embryonic period in which the embryo remains spherical, which facilitates embryo collection, and the possibility of repeated embryo collections from the same animals over successive estrus cycles. Additionally, the stallion serves as an excellent model for the human male, as stallions are typically not selected for sperm quality nor the ability of semen to be cryopreserved, in contrast to males in production species, such as the bull. Moreover, since many stallions reach advanced age, the horse can be used as a model to study the impact of paternal age on embryo quality.

    Our study, focused on three embryo ages (8, 10 and 12 days post ovulation), revealed a significant impact of sperm cryopreservation on the transcriptome of the resulting embryo. Importantly, transcripts with decreased abundance reflected genes related to DNA replication and assembly, and oxidative phosphorylation. Exploration of differentially-expressed genes at the molecular and cellular level revealed alterations in important functions including ATP synthesis, regulation of transcription, nucleosome assembly, chromatin silencing, protein synthesis, and redox regulation. Alterations in these genes help to explain the reduced fertility observed with cryopreserved sperm attributable to increased early embryo mortality [11, 12].

    The pre-implantation period is a period of rapid embryo growth, requiring a ready supply of ATP. The equine embryo appears to have a significant capacity for glycolysis, but also uses oxidative phosphorylation [36]. The KEGG pathways analysis of downregulated genes revealed enriched annotations for oxidative phosphorylation, pyruvate metabolism, glycolysis, and the TCA cycle, suggesting compromised energy metabolism in CRYO embryos. A similar picture was observed in Day-10 and Day-12 embryos, with the pathways for oxidative phosphorylation, metabolic pathways, and non alcoholic fatty liver disease significantly over-represented in transcripts with reduced abundance of all CRYO embryos obtained.

    When we evaluated low-abundance equine transcripts for their mouse orthologs, we found that many of the genes downregulated in CRYO embryos have knockout database annotation terms related to reduced embryonic viability. This finding opens the possibility that not only genes related to the metabolism and thus growth of embryos, but also genes directly related to embryo organogenesis, embryo survival, and offspring health are affected by the use of cryopreserved sperm, and thus these genes warrant further investigation.

    While the mechanisms behind the effects reported here are as yet unclear, a major factor may be the well-documented oxidative damage that the genome and epigenome experiences during cryopreservation and thawing [1114]. Cryopreservation is a major cause of oxidative stress [37] and lipid peroxidation in stallion spermatozoa [10, 17, 38, 39]. Lipid peroxidation in spermatozoa surviving cryopreservation [37] is associated with increased levels of 4-hydroxinonenal (4-HNE) [17]. This compound is able to interact with DNA to form adducts that have been related directly to increased rates of mutation in important cell-cycle regulators [40, 41]. The production of 4-HNE during cryopreservation of stallion spermatozoa is well documented [10, 17, 39], and it is possible that significant amounts of 4-HNE and other toxic lipid aldehydes are incorporated to the oocyte, potentially causing alterations in embryo development. In addition to DNA damage, 4-HNE can alkylate the sperm centrioles, and in horses, as in humans, paternal centrioles are inherited by the embryos. Damaged centrioles may cause disrupted cytoskeletal protein organization during early cleavage [42].

    Supporting this line of reasoning, recent reports have linked abnormal early cleavage events and changes in embryo transcript abundance to fertilization with spermatozoa showing oxidative stress. Macaque embryos obtained after fertilization with ROS-treated sperm showed significantly lower rates of development to the four- and eight-cell stages, and changes in transcript abundance for genes related to actin cytoskeleton organization, cell junction assembly and cell adhesion [43]. Although seen at a much later stage of development, in our study we also found that genes for cytoskeleton components tubulin alpha 1 a, tubulin beta 2 class II a and actin, cytoplasmic 1, N-terminally processed were downregulated in 8-day CRYO embryos.

    Cryopreservation may also directly affect the epigenome of the paternal DNA; recent studies have shown that cryopreservation increases the level of DNA methylation in equine sperm [12] and the expression of genes important to intracellular regulation of epigenetic status [16]. Notably, we also found significant reduction in abundance of transcripts for histones in CRYO embryos.

    The finding that many differentially regulated genes in CRYO embryos are orthologs of mouse genes that have knockout database annotation terms related to reduced embryonic viability provides further evidence linking cryopreserved sperm to reduced embryonic viability. These annotations consistently appeared on analysis of low-abundance transcripts in all CRYO embryos, and included genes related to embryonic growth retardation and embryo lethality. Interestingly, annotations related to male and female infertility were also present; this warrants further investigation on the effect of sperm origin on the fertility of resulting offspring.

    In summary, the present study provides for the first time transcriptomic analysis of equine embryos in relation to the handling of semen used for their production, however we acknowledge the preliminary and descriptive nature of this report but our data provide strong evidence that cryopreservation of sperm exerts a profound impact on the transcriptome of early embryos. Our findings may stimulate new lines of research to improve this biotechnology in humans and animals.
    Last edited by vineyridge; Jun. 29, 2019, 01:51 PM.
    "I'm a lumberjack, and I'm okay."
    Thread killer Extraordinaire

  • #2
    Very interesting! Does this mainly affect the viability of the embryo, or are there health consequences for the foal/horse?

    To me it reads like the main question is viability of the embryo, but am I missing something?


    • #3
      Those are my questions as well. Hopefully that means that the embryo is more likely to fail when created with frozen but if carried to term will go on to be the same horse as they would have been if fresh semen had been used.
      McDowell Racing Stables

      Home Away From Home


      • Original Poster

        The article does say this:

        Moreover, appreciation of the contribution of sperm to embryo development has evolved from the concept that the only role of sperm at fertilization is to introduce the male genome into the egg. Sperm carry a myriad of small noncoding RNAs with potential roles in early embryo development [21, 22]. Notably, sperm carry the activating factor PLCζ, which triggers calcium oscillations that induce oocyte activation [23, 24], and it has been shown in mouse and rabbit that alterations in frequency and amplitude of post-fertilization calcium oscillations can affect the phenotype of the resulting embryo into post-implantation development and adulthood [25, 26]. Thus, there are extensive pathways by which cryopreservation of sperm could alter the development of the fertilized oocyte and embryo.
        So at this point in time it would seem to be an open question whether a foal is different if produced by frozen semen.
        "I'm a lumberjack, and I'm okay."
        Thread killer Extraordinaire


        • #5
          Sooooooo cool, I can't wait to read the whole article. Thank you!!!!!!!!!
          Proud member of the Colbert Dressage Nation